Glycogen Synthase Kinase 3 beta confers refractoriness to the microRNA-210-elicited exacerbation of hypoxia-reoxygenation stress-induced apoptotic cell death

Abstract

Abstract Introduction Apoptotic cell death is a detrimental signatory molecular hallmark of Ischemia / Reperfusion (I/R) injury. Despite the exhaustive findings and significant advancements in the realm of I/R research, there is a paucity of viable molecular strategies that could be targeted to combat the underlying deleterious biochemical pathways that precipitate Hypoxia-Reoxygenation (H-R) stress, the cellular manifestation of I/R injury. The master hypoxamiR, microRNA-210 (miR-210), is an indispensable molecular component of the protective cellular adaptive response to hypoxia stress that paradoxically exacerbates apoptotic cell death during the cellular reoxygenation phase. While the entire repertoire of molecular entities mediating this paradoxical deleterious impact of miR-210 on H-R stress induced apoptotic cell death are largely unknown, recent studies have unveiled and posited, the evolutionary conserved serine/threonine kinase, Glycogen Synthase Kinase 3 beta (GSK3β) as a pivotal molecular focal point of the convergent and divergent signaling cascades that modulate the miR-210-regulated apoptotic cell death during the cellular reoxygenation phase. Purpose In this study, we elucidated and delineated the role of GSK3β (in)activation in the miR-210-induced exacerbation of the H-R stress-induced apoptotic cell death in AC-16 cardiomyocytes. Methods Using transient overexpression vectors and decoy / inhibition vectors to modulate miR-210 expression concomitant with the ectopic expression of the constitutive active GSK3β S9A (ca-GSK3β S9A) and the catalytically-dead dominant negative GSK3β K85A (dn-GSK3β K85A) mutants, we performed exhaustive biochemical and molecular assays determining apoptotic cell death in AC-16 cardiomyocytes subjected H-R stress. Results Caspase-3 activity analysis coupled with DNA fragmentation assays demonstrated that the H-R driven augmented GSK3β kinase activity significantly attenuates the miR-210-induced exacerbation of H-R evoked apoptotic cell death during the cellular reoxygenation phase. Resolution of the multimodal components of the apoptotic signaling pathway, unveiled that the H-R driven augmented GSK3β kinase activity modestly increases the miR-210-driven exacerbation of the intrinsic apoptotic pathway during the cellular reoxygenation phase, as molecularly inferred by the levels of the release of Cytochrome C from the mitochondria into the cytosol. However, in a stark contrast, H-R driven augmented GSK3β kinase activity concomitantly overwhelmingly attenuated the flux through the extrinsic apoptotic pathway during the cellular reoxygenation phase, as molecularly inferred by caspase-8 activity analysis. Conclusion Our study is the first to unveil that the H-R driven augmented GSK3β kinase activity during the cellular reoxygenation phase, is an inherent protective cellular adaptive response that mitigates the miR-210-orchestrated exacerbation of the cellular reoxygenation-elicited apoptotic cell death.